Throughout the specification, there are several noted references. The citations for those references are provided below:
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Chong, S. S. C., and Vielkind J. R. (1989) Expression and fate of CAT reporter gene microinjected into fertilized medaka (Oryazias latipes) eggs in the form of plasmid DNA, recombinant phage particles and its DNA. Theor. Appl. Genet. 78: 369-380
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Davies, P. L., Fletcher, G. L. and Hew, C. L. (1989) Fish antifreeze protein genes and their use in transgenic studies. in: Oxford Surveys on Eukaryotic Genes, 6: 85-110. Edited by Norman Maclean, Published by Oxford University Press.
Davies, P. L. and Hew, C. L. (1990) Biochemistry of fish antifreeze proteins. The FASEB Journal, 4:2640-2648.
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Fletcher, G. L., Idler, D. R., Vaisius, A. and Hew, C. L. (1989) Hormonal regulation of antifreeze protein expression in winter flounder. Fish Physiology and Biochemistry, 7:387-393.
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A variety of attempts and successes have been made to develop transgenic fish and other animals. Growth hormone has been of particular interest. Growth hormone is a single chain polypeptide hormone that plays a principal role in the regulation of somatic growth and development in animals. Many approaches have been made to increase fish growth by growth hormone. These include the feeding of pituitary extracts (Tucker 1936, Cantilo and Regalado 1940), injection or implantation of purified recombinant-derived growth hormone (Gill et al. 1985, Sekine et al. 1985, Kawauchi et al. 1986, Agellon et al. 1988). All these results clearly showed that growth hormone alone is effective in stimulating fish growth. However, all these studies have limited application in aquaculture, and one major drawback is that the phenotype can not be inherited.
Gene transfer technique has become a new and powerful approach to manipulate the genetic and phenotypic characteristic of both animals and plants. Various reports have been made in the production of transgenic fish. The first transgenic study on fish was reported by Vielkind et al. (1982). These investigators injected swordtail tumour genes into the Platyfish, and found that the injected swordtail Tu genes could induce T-melanophore induction in Tu-free Platyfish. In 1985 and 1986, Zhu et al. reported the production of transgenic fish by growth hormone gene transfer. Using a mouse metallothionein promoter ligated to a human GH structural gene, they successfully produced transgenic loach, goldfish and silver carp. On the average, the transgenic fish was 1 to 3 times larger than control. Since then, several reports using similar gene constructs have been published (Rokkones et al. 1989, Guyomarde et al. 1989, Chen et al. 1990)
Most, if not all of these studies were carried out by using either mammalian GH or mammalian gene and viral promoters. To be acceptable in aquaculture, the promoter(s) and gene(s) used in transgenic fish should be derived preferably from fish protein genes without posing any potential health hazards. Furthermore the production of a strain of faster growing fish in an economically important species such as salmonids with an all fish gene construct will be beneficial to fish farming.
We have discovered the successful production of transgenic Atlantic salmon by using a fish gene promoter derived from ocean pout antifreeze gene (OP-AFP), (Hew et al. 1988) and the GH cDNA gene from chinook salmon (Hew et al. 1989).
According to an aspect of the invention, a promoter for use in constructing a chimeric gene construct is provided. The promoter comprises an antifreeze gene (AFP) promoter having characteristics functionally corresponding to the AFP promoter derived from Ocean pout and 3' sequence containing the normal RNA transcriptional termination signal. The promoter is characterized by a 2 kb Bam H1 - B1 II fragment of Op-AFP of FIGS. 1a and 1b and Table I. The 3' sequence is characterized by the 1 kb HpaI - Hind III fragment in FIG. 1b. Functional analysis of other antifreeze promoters, including wolffish (WO), sea raven (SR) and winter flounder (WF), shows that they can be used in a similar fashion (Table II). Here only the Ocean pout antifreeze protein promoter is used as a teaching example in producing transgenic fish.
The invention provides specific embodiments, such as the analysis of transient expression of the OP-AFP gene promoter activities in a salmonid cell line and the Japanese medaka embryos, the construction of AFP-GH fusion gene, and its gene transfer by microinjection, screening of transgenic salmon by polymerase chain reaction (PCR), size and growth rate measurements.